Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling

Pseudouridine (Psi) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ts in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D-2. The pseudouridylation reaction in those cells results in the exchange of the D at the CS of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Psi s. We present here the experimental details of this method and show that it allows the identification of all Psi s in human major nuclear and nucleolar RNAs, including several previously unknown Psi s. Because the method allows direct determination of Psi s at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.